WO2011083933A2 - Mutant strain for producing l-ornithine or l-arginine, and method for producing same - Google Patents

Abstract

The present invention relates to a polynucleotide that is active to an acetyl glutamate synthase and acetyl ornithinase which are associated with ornithine or arginine biosynthesis from Corynebacterium glutamicum. The present invention also relates to a polypeptide encoded by said polynucleotide, a recombinant vector comprising said polynucleotide, to a transformant obtained by introducing said recombinant vector to a host microorganism for producing L-ornithine or L-arginine, and transforming the recombinant vector, and to a method for producing L-ornithine or L-arginine by culturing said transformant. The activity of the transformant of the present invention to an acetyl glutamate synthase and acetyl ornithinase is increased as compared to an intrinsic activity, and thus L-ornithine or L-arginine can be produced, at a high yield rate, from the transformant of the present invention.

Description

L-ornithine or L-arginine production mutant strain and preparation method thereof

The present invention relates to L-ornithine or L-arginine production mutant strains and a method for preparing the same, and more specifically, to acetylglutamate synthase and acetylornithinase related to ornithine or arginine biosynthesis in Corynebacterium glutamicum. A polynucleotide having an activity, a polypeptide encoded by the polynucleotide, a recombinant vector comprising the polynucleotide, a transformant transformed by introducing the recombinant vector into an L-arginine producing host microorganism, and culturing the transformant To produce L-ornithine or L-arginine.

L-amino acids are used in human medicine, especially in the pharmaceutical industry, food industry and animal nutrition. In particular, L-ornithine in L-amino acids is known as a pharmaceutical ingredient that promotes liver function as an intermediate of arginine biosynthesis (Salvatore et al., 1964). In addition, L-arginine is freely contained in plant seeds and garlic, and is also used as an amino acid-enhancing agent and widely used in medicine and food. It is used in medicine for liver function promoter, brain function enhancer, male infertility drug, synthetic amino acid preparation, etc., and for food, fish jelly additive, health drink additive, and salt substitute for hypertension patients have recently been spotlighted.

Coryneform bacteria, especially corynebacterium glutamicum, can produce amino acids by fermentation of strains. Because of its great importance, the production method has been continuously improved, and for example, there has been a methodological improvement in culture medium composition such as stirring and oxygen introduction or sugar concentration during fermentation.

To increase the amino acid productivity of these microorganisms, microbial screening and variant screening methods are used. In this way, strains are obtained that are nutritionally demanding for metabolites that are resistant to, or regulated by, anti-metabolites and produce L-amino acids. For example, the glutamic acid method of producing (glutamate) producing strain of Brevibacterium (Brevibacterium) or Corynebacterium (Corynebacterium) tin ornithine by using a mutant strain derived from a microorganism of the genus (EP 0 393 708 A3) are reported It became. Further, the carbon source, there is provided a method of producing L- arginine directly from nitrogen source, glutamic acid (glutamate) producing strain of Brevibacterium (Brevibacterium) or Corynebacterium, a method using a mutant strain derived from the (Corynebacterium) in microorganisms (Japanese Published Unexamined Patent digests 57-163487, 60-83593, 62-265988).

Recombinant DNA technology is a method that is further used to improve the inherent properties of L-amino acid producing strains of Corynebacterium. For example, there is a method using a strain amplified ornithine biosynthetic gene argCJBD in a strain that can not synthesize arginine and proline (Hwang et al., 2008). In addition, a method using a recombinant strain in which the gene argR that suppresses the expression of arginine biosynthetic operon is used (US Patent Application No. 2002 / 0045223A1) has been reported.

On the other hand, improved production capacity is also achieved by strengthening the biosynthetic genes. It is known that the yield can be improved by increased expression of biosynthetic genes. For example, a method of over-expressing argF , which is an arginine biosynthesis gene (Korean Patent Application No. 10-2004-107215), has been reported.

In addition, L-arginine can be produced at high yield by overexpressing the argD2 gene (Ncgl2355) or the gene (Ncgl0990), which are presumed to be the genes of acetylornithine amino group transferase related to arginine biosynthesis in Corynebacterium glutamicum strains. A method for producing arginine is disclosed (Korean Patent Nos. 10-0830289 and 10-0830290).

In order to make high yields of ornithine or arginine producers, the biosynthetic genes need to be strengthened, and in particular, the biosynthetic enzymes, acetylglutamate synthase and acetylornithinase, need to be strengthened.

However, since the acetylglutamate synthase gene has not been known to date in microorganisms of Corynebacterium, the present inventors have identified a gene that performs the function of acetylglutamate synthase. At the same time, it was confirmed that this gene has similar activity to acetylornithase encoded by argJ . In addition, the present invention was completed by confirming that the concentration of ornithine or arginine is improved by enhancing the present gene.

It is an object of the present invention to novelly identify polypeptides or polynucleotides having the activity of acetylglutamate synthase or acetylornithase from the genus Corynebacteria.

In addition, another object of the present invention is to provide a strain with improved expression of ornithine or arginine by overexpressing the gene.

Still another object of the present invention is to provide a method for producing ornithine or arginine at high concentration using the microorganism.

In order to achieve the above object, one aspect of the present invention is to provide a polypeptide having the activity of acetyl glutamate synthase and acetyl ornitinase derived from Corynebacterium glutamicum.

In the present invention, the gene number Ncgl1469 deposited in the "National Insititute of Health" which is a publicly accessible database for identifying genes encoding acetylglutamate synthase and acetylornithase encodes acetylglutamate synthase and acetylornithase. It was confirmed that. There is no known gene encoding acetylglutamate, and acetylornithase has been reported to be encoded by argJ (Sakayan et al., 1996). However, reports of the Ncgl1469 gene encoding two enzymes were previously unknown. The amino acid sequence is listed in SEQ ID NO: 1, and the nucleotide sequence is listed in SEQ ID NO: 2.

Another aspect of the invention is to provide a vector comprising a gene encoding acetylglutamate synthase and acetylornithase. The vector comprises an acetylglutamate synthase and an acetylornithase gene. Plasmids that can be used above include pZ1 (menkel et al), pEkEx1 (Eikmarms et al), pHS2-1 (Sonnen et al), pCG4 (US pat. No. 4,489,190) or pNG2 (Serwold-Davis et al) and pEKO (Eikmanns et al). Preferably pEKO is used in the present invention. More preferably the vector comprises an amino acid of SEQ ID NO: 1 or a nucleotide of SEQ ID NO: 2. Specifically, the vector shown in FIG. 1 may be mentioned.

Another aspect of the invention is to provide a microorganism or transfected cell or recombinant cell enriched in acetylglutamate synthase and acetylornithase. The strains used may employ strains that already produce L-ornithine or L-arginine prior to enrichment of acetylglutamate synthase and acetylornithase. The transformant can be easily prepared by those skilled in the art according to any transformation method. In the present invention, "transformation" refers to a phenomenon in which DNA is introduced into a host so that DNA can be reproduced as a factor of a chromosome or by chromosome integration, thereby causing an artificial genetic change by introducing an external DNA into a cell. Means.

In general, the Hanahan method, the electroporation method, the calcium phosphate precipitation method, the protoplast fusion method, and silicon carbide, were used for the transformation method by using CaCl ₂ precipitation method and CaCl ₂ method using a reducing material called DMSO (dimethyl sulfoxide). Agitation with fibers, agro bacteria mediated transformation, transformation with PEG, dextran sulfate, lipofectamine and dry / inhibition mediated transformation.

Specifically, in the present invention, a transformant was prepared by introducing the recombinant vector pEK-Ptrc :: 1469 into a host microorganism using electroporation, and a strain containing the recombinant vector was isolated using antibiotic resistance.

In the present invention, the concept of "enhancement rather than intrinsic activity" means that the intracellular activities of the above-mentioned enzymes acetylglutamate synthase and acetylornithase are improved compared to those naturally occurring. The method for increasing the activity of the enzyme may be to increase the number of gene copies. To increase the number of copies of a gene, a method using a vector such as a plasmid or inserting an additional gene into a chromosome may be used. It can also be further enhanced by enhancing regulatory factors that positively affect gene expression. Regulatory elements can thus be enhanced at the level of transcription, particularly for enhanced transcriptional signals. In addition to the above, translation can also be enhanced, thereby improving the stability of, for example, mRNA. In addition, a gene encoded by a corresponding enzyme having high activity may be used. Overexpression of the genes of interest can also be reached by changing the medium composition. Another way could be to enhance the promoter or to replace the stronger promoter. What can be used as a strong promoter can be used, for example, the Tac-promoter (Amann et al). Another way to increase the activity of the enzyme is to increase the endogenous activity in the microorganism through mutation. Mutations of this kind can be generated, for example, by traditional methods such as UV rays or mutational mitigation chemicals or by genetic engineering methods such as deletions, insertions and / or nucleotide exchanges. In order to enhance the activity of the enzyme it is possible to combine all the above-mentioned measures possible.

According to the invention, Coryne or Brevi bacterium can be used among Coryneform bacteria, and specifically suitable starting strains of Corynebacterium glutamicum type are, for example, the following known wild type strains. Corynebacterium glutamicum ATCC13032, Corynebacterium acetoglutamicum ATCC15806, Corynebacterium acetoacidophilum ATCC13870, Corynebacterium bacteriumium thermoaminogenaes) FERM BP-1539, Brevibacterium flavum ATCC14067, Brevibacterium lactofermentum ATCC13869, Brevibacterium divaricatum ATCC14020 and from them Deficient ornithine carbamoyltransferase deficiency involved in chitin biosynthesis pathway, deficiency in arginine repressor and variant producing glutamate kinase or deficiency in arginine repressor involved in arginine biosynthesis Number of variants that produce excess L-arginine at improved concentrations have.

In another embodiment of the invention, the microorganisms described above can be cultured for the purpose of production of L-ornithine or L-arginine. Cultivation process of the microorganism may be made according to the appropriate medium and culture conditions known in the art. This culture process can be used by those skilled in the art can be easily adjusted according to the strain selected. Examples of the culture method include, but are not limited to, batch culture, cintinuous culture, and fed-batch culture. Such various culture methods are disclosed, for example, in "Biochemical Engineering" (James M. Lee, Prentice-Hall International Editions, pp 138-176, 1991). During the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. In addition, during the culture, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble generation. In addition, in order to maintain the aerobic condition of the culture, oxygen or an oxygen-containing gas (eg, air) is injected into the culture. The temperature of the culture is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. The incubation period can continue until the desired amount of L-arginine methionine production is obtained, preferably 10 to 160 hours. Isolation of L-ornithine or L-arginine from the culture can be separated by conventional methods known in the art. In such a separation method, methods such as centrifugation, filtration, ion exchange chromatography, and crystallization may be used. For example, the supernatant obtained by removing the biomass by centrifugation of the culture at low speed can be separated by ion exchange chromatography.

As described above, the present invention relates to a polynucleotide having activity on acetylglutamate synthase and acetylornithase related to ornithine or arginine biosynthesis in Corynebacterium glutamicum, a polypeptide encoded by the polynucleotide, the polynucleotide Recombinant vector comprising a transformant transformed by introducing the recombinant vector into the L-arginine producing host microorganism and the effect of providing a method for producing L- ornithine or L-arginine by culturing the transformant have. The transformant of the present invention is capable of producing L-ornithine or L-arginine in high yield by enhancing the activity of acetylglutamate synthase and acetylornithase than intrinsic activity, and thus have a useful effect in the biopharmaceutical industry. have.

1 is a diagram showing the structure of a pEK-Ptrc :: Ncgl1469 vector according to the present invention.

Below. The present invention will be described in more detail with reference to Examples. However, these examples are only for carrying out the present invention by way of example, but the scope of the present invention is not limited to these examples.

<Example>

Example 1: Ncgl1469 Cloning

In this example, PCR was performed using a trc promoter as a template of pTrc99A (Amann et al. 1988) and oligonucleotides of SEQ ID NOs: 3 and 4 as primers to prepare an overexpression vector. In addition, PCR was performed using rrnB terminator as a template of pTrc99A and oligonucleotides of SEQ ID NO: 5 and 6 as primers. The polymerase is PfuUltra ^™ high-trust DNA polymerase (stratagene) and PCR conditions were denatured 95 ° C., 30 seconds; Annealing 55 ° C., 30 seconds; And 25 degreeC of the polymerization reaction 68 degreeC was repeated 1 time. Base sequence information of the NCgl1469 gene was obtained based on NIH GenBank, and primers were synthesized based on this (SEQ ID NOS: 7 and 8). Using the synthesized primers, PCR is performed in the same manner as above using the gene DNA of the Corynebacterium glutamicum ATCC13032 strain as a template. PCR conditions were denatured 95 ℃, 30 seconds; Annealing 55 ° C., 30 seconds; And 25 minutes of polymerization reaction at 68 ° C. for 1 minute 30 seconds.

The pEKO (E. coli-C.glutamicum shuttle vector, Eikmanns et al. 1991) vector was subjected to restriction enzyme treatment with XbaI. The ptrc promoter fraction and 1469 ORF obtained above were subjected to restriction enzyme treatment with Xba1, NdeI / NedI, and xba1, respectively, and then three-piece conjugation with the enzyme-treated vector to obtain a recombinant vector. The obtained recombinant vector was enzymatically treated with HincII and EcoRI again, and the rrnB terminator fraction obtained by PCR was treated with SmaI / EcoRI restriction enzyme to obtain pEK-Ptrc :: 1469 recombinant vector with promoter-1469 ORF-terminator inserted. It was.

Example 2 Ncgl1469 Overexpression

Ncgl1469 cloned in Example 1 In order to confirm the activity of the gene, it is necessary to prepare a strain argJ crushed of Corynebacterium glutamicum ATCC13032 strain first. The pK18mobsacB integration vector (Schafer et al. 1994) was used to prepare a strain lacking argJ. PCR was performed using ATCC13032 as a template and SEQ ID NOs. 7 and 8, 9 and 10 as primers, respectively. PCR conditions were denatured 95 ℃, 30 seconds; Annealing 55 ° C., 30 seconds; And 25 minutes of polymerization reaction at 68 ° C. for 1 minute 30 seconds. After restriction enzyme treatment of the pK18mobsacB vector with XbaI, the two PCR fractions were treated with XbaI / ApaI and ApaI / XbaI, respectively, to construct a recombinant vector by three-piece conjugation.

The recombinant vector was transformed into ATCC13032 strain by electric pulse method, and then, the strain inserted by homology with nucleotides on the chromosome was selected in a selection medium containing 25 mg / L of kanamicin. The primary chromosome inserted strains were shaken in nutrient medium (30 ° C., 4 hours), and then plated on solid medium containing sucrose from 10 ⁻⁴ to 10 ⁻¹⁰ , respectively. By selecting the colonies where most colonies were killed by sucrose and appearing at a low rate, strains from which vector sequences on the chromosomes inserted by secondary crossover were removed were selected. The strains selected as described above were finally selected through the gene structure confirmation process through confirmation of susceptibility to the antibiotic kanamycin and PCR using SEQ ID NOS: 7 and 10.

Example 3: Determination of Acetylglutamate synthase and acetylornithase activity of Ncgl1469

After culturing the strain selected in Example 2 in a liquid medium, the cells were collected by centrifugation. The cells were prepared by washing twice in 100 mM Tris / Hcl buffer (pH 7.5), and cell walls were removed by the method using glass beads.

According to a known method, acetylglutamate activity was measured by measuring the amount of 5-thio-2-nitrobanzonate formed at 412 nm using a spectrophotometer (Errey and Blanchard, 2005). The activity of acetylornithase was also measured by conventional methods (Vogel and Mcleelan, 1970).

Through the above method, the results of measuring the activity of the strain overexpressing the pEKO vector and pEK-Ptrc :: 1469 in the ATCC13032 and argJΔ strains are shown in Table 1 below.

Table 1

Strain	Plasmid	Specific activity (units / mg protein) *
		Acetylglutamate synthase a	Acetyl ortase b
C. glutamicum argJ	pEK0	0.03	ND
	pEK-P trc :: 1469	0.17	0.07

ArgJ-deficient strains reported to encode acetylornithase were prepared to overexpress Ncgl1469, and acetylglutamate and acetylornithase activity were measured to confirm that Ncgl1469 had activity against both enzymes.

Example 4: Ornithine Production of SJ8073-pEK-Ptrc :: 1469 Strain

In order to confirm ornithine production of the overexpressed strain Ncgl1469 gene ornithine strain SJ8074 (argF-argR-proBΔ, Hwang et al. 2008) strain was used as a parent strain.

Strains 0.8 g / L KH ₂ PO ₄ , 10 g / L (NH ₄ ) ₂ SO ₄ , 1 g / L MgSO ₄ · 7H ₂ O, 1.2 g / L Na ₂ HPO ₄ , 2 mg / L MnSO ₄ · H ₂ O, 10 mg / L ZnSO ₄ · 7H ₂ O, 10 g yeast extract, 20 g / L CaCO ₃ , 60 g / L glucose and 10 mM IPTG was added to shake culture in a 250 mL baffle flask. Ornithine concentration in the culture is shown in Table 2 below.

TABLE 2

Strain	Plasmid	l-ornithine concentration (mg / l) a
		Glutamate (0 mM)	Glutamate (50 mM)
C. glutamicum SJ8074	pEK0	137.57	145.27
	pEK-P trc :: 1469	178.81	207.84

As a result of overexpressing Ncgl1469 in SJ8074, an ornithine-producing strain, ornithine production was increased by more than 20%.

Recombinant vector pEK-Ptrc :: Ncgl1469 to C. The transformant obtained by introducing into glutamicum SJ8074 strain was named Corynebacterium glutamicum CA06-0020, and was assigned to the Korean Culture Center of Microorganisms (hereinafter abbreviated as "KCCM") as of December 23, 2009. Deposited with KCCM 11057P.

Example 5: Arginine Production of ATCC21831, argR Δ -pEK-Ptrc :: 1469 Strain

In order to confirm the arginine production capacity of the strain overexpressing the TL2 gene, a strain was prepared by crushing argR, which is an arginine inhibitor, of the ATCC21831 strain, which is an arginine producing strain, and used as a parent strain.

Strains 0.8 g / L KH ₂ PO ₄ , 10 g / L (NH ₄ ) ₂ SO ₄ , 1 g / L MgSO ₄ · 7H ₂ O, 1.2 g / L Na ₂ HPO ₄ , 2 mg / L MnSO ₄ · H ₂ O, 10 mg / L ZnSO ₄ · 7H ₂ O, 10 g yeast extract, 20 g / L CaCO ₃ and 60 g / L glucose are added and shake incubated in a 250 mL baffled flask. Arginine concentration in the culture is shown in Table 3 below.

TABLE 3

Strain	Plasmid	l-arginine concentration (mg / l) a
		IPTG (0 mM)	IPTG (10 mM)
ATCC21831 argRΔ	pEK0	1.42	1.39
	pEK-P trc :: 1469	1.39	1.56

As a result of overexpressing Ncgl1469 in the ATCC21831-argRΔ strain, which is an arginine producing strain, it was confirmed that the arginine production was increased by about 10%.

The transformant obtained by introducing the recombinant vector pEK-Ptrc :: Ncgl1469 into the ATCC21831 argRΔ strain was named Corynebacterium glutamicum CA06-0021, and as of December 23, 2009, the Korean Culture Center of Microorganisms, hereinafter, " KCCM 11058P (abbreviated as "KCCM").

From the above description, those skilled in the art can understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the examples and experimental examples described above are exemplary in all respects and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims and the equivalent concept described below are included in the scope of the present invention rather than the foregoing detailed description.

As described above, the present invention relates to a polynucleotide having activity on acetylglutamate synthase and acetylornithase related to ornithine or arginine biosynthesis in Corynebacterium glutamicum, a polypeptide encoded by the polynucleotide, the polynucleotide Recombinant vector comprising a, transformant transformed by introducing the recombinant vector into the L-arginine producing host microorganism and the effect of providing a method for producing L- ornithine or L-arginine by culturing the transformant have. The transformant of the present invention is capable of producing L-ornithine or L-arginine in high yield by enhancing the activity of acetylglutamate synthase and acetylornithase than intrinsic activity, and thus have a useful effect in the biopharmaceutical industry. have.

Claims (7)

1. A polypeptide represented by amino acid sequence 1 having the activities of Corynebacterium glutamicum-derived acetylglutamate synthase and acetylornithase.
2. A polynucleotide represented by SEQ ID NO: 2 encoding the polypeptide of claim 1.
3. Recombinant vector pEK-Ptrc :: Ncgl1469 of FIG. 1 comprising the nucleotide of claim 2.
4. A microorganism of the genus Corynebacterium transformed with the vector of claim 3, wherein the activity of acetylglutamate synthase and acetylornithase has an enhanced amino acid production ability than the intrinsic activity.
5. 5. The microorganism of the genus Corynebacterium according to claim 4, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.
6. 5. The microorganism of the genus Corynebacterium according to claim 4, wherein the amino acid is L-ornithine or L-arginine.
7. A method for preparing L-ornithine or L-arginine, comprising culturing the transformed Corynebacterium glutamicum genus microorganism of claim 4 and recovering ornithine or arginine from the microorganism.

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